(a) Field of the Invention
The present invention relates to an expression vector for animal cells, and more particularly, to an expression vector including a nuclear matrix attachment region element (hereinafter referred to as “MAR element”) and the transcription termination site of the gastrin gene.
(b) Description of the Related Art
Many kinds of expression systems, such as microorganisms, plants, yeasts, insect cells, and animal cells are currently in use for medical treatment through expression of the desired proteins in a large amount. Among the many kinds of expression systems, microorganisms are most easily used, and many kinds of microorganism systems are studied and utilized as expression systems.
However, the use of a microorganism expression system is limited in some respects. First, though genes are expressed, the structure and characteristics of an expressed protein are unlike that of an animal protein because a microorganism has a different mechanism for expressing proteins and for modifying protein by glycosylation, phosphorylation, and amidylation. Therefore, a recombinant protein produced by microorganisms has nearly no modification, and is limited to the production of proteins whose functions are not much affected by the differences in the modification and structure of the proteins. In addition, when the recombinant proteins expressed by microorganisms are used, a cleaning process for contamination by the microorganism or a toxin is needed.
Although animal cells are suitable for an animal's protein expression, an expression system using animal cells is not commonly used because the expression system using animal cells creates higher production costs due to a lower expression efficiency of recombinant protein compared to microorganisms.
Animal cells currently in use in industry as an expression system include. CHO (Chinese Hamster Ovary), BHK (Baby Hamster Kidney), and myeloma cells. An expression vector is introduced into an animal cell and a desired foreign protein is produced, similar to a microorganism.
Gene expression systems are modified by various methods, because generally a small amount of the foreign genes are expressed. For example, a cell strain of CHO cells is cultivated in medium containing methotrexate (hereinafter referred to as “MTX”) which is an inhibitor of DHFR (Dihydrofolate reductase), in order to obtain a CHO strain which is alive, depending on MTX concentration, and highly expresses protein due to an increase in the copy number of genes.
Generally, when foreign genes are expressed in an animal cell, the foreign gene is co-transfected with a vector having a selective marker, and transformed cells are selected through cultivation in selective medium for many hours. However, the frequency of achieving a highly expressing cell clone is low. The low frequency of foreign gene expression is due to chromosomal insertion of the foreign gene in the animal system, unlike a microorganism. In addition, though the insertion process of the foreign gene is successful, the expression of the foreign gene cannot be expected since the inserted site of each gene differs and the expression of a gene depends on the inserted site (Grindley et al., 1987. Trends Genet. 3, 16–22; Kucherlapati et al., 1984. Crit. Rev. Biochem. 16, 349–381; Palmiter et al., 1986. Annu. Rev. Genet. 20, 465–499). Therefore, though foreign genes are stably integrated, they may be expressed in small amounts because most of the gene expression in animal cells is inhibited by the neighboring nucleic acid (Eissenberg et al., 1991. Trends Genet. 7,335–340; Palmiter et al., 1986. Annu Rev. Genet. 20, 465–499).
In order to protect the expression of the foreign gene from position effects, the possibility of using nuclear matrix elements in several systems has been reported. An exemplary nuclear element includes an insulator element, nuclear matrix attachment region (hereinafter referred to as “MAR”), and a scaffold attachment region (hereinafter referred to as “SAR”).
Kalos (Kalos et al., 1995 Mol. Cell. Biol. 15, 198–207) suggested that when apolipoprotein B MAR combined with a minimal promoter transgene construct, the foreign gene was stably introduced in the host chromosome, so that the expressed amount of the transcript increased by about 200 times. Similar to the aforementioned method, it was reported that chicken lysozyme A MAR and β-interferon SAR are capable of increasing the expression level of a foreign gene in a vertebrate cell regardless of the chromosomal insertion site (Eissenberg et al., 1991. Trends Genet. 7, 335–340; Klehr et al, 1991. Biochemistry 30, 1264–1270). However, it has not been verified that the MAR and SAR are capable of increasing protein production in a CHO cell strain, or that the MAR and SAR are suitable for common use.
When an animal cell gene is expressed, mRNA synthesis occurs from the promoter and stops at the termination site. The levels of the expressed proteins are often influenced by the efficiency of transcription termination as well as the stability of the synthesized mRNA.
The transcriptional termination site which is included in an expression vector controls polyadenylation, and has an influence on mRNA stability. The termination site includes a poly-A signal, cleavage site, and termination site; the polyadenylation signal is AATAAA and is well studied. However, the cleavage site where polyadenylation occurs, and the termination site where the gene transcription is completed by RNA polymerase enzyme II are not well known. In addition, though it is reported that GU/U-rich region except the three kinds of critical region controls the polyadenylation of mRNA, the detailed mechanism is not known.
Expression vectors which are commonly used in animal cells contain a poly-A signal of SV40 virus and BGH (Bovine Growth Hormone). It has not been suggested that a specific terminator that improves mRNA stability and the expression level be developed in order to use an expression vector in animal cells.